The overriding goal of this proposal is to understand the role of molecular chaperones in regulating the function of steroid receptors. The Specific Aims of this proposal seek to define the mechanisms responsible for ordered, dynamic chaperone interactions with steroid receptors. Key to these ordered interactions are partner proteins-Hip, Hop, and BAG1 - for the major nucleocytoplasmic chaperones Hsp70 and Hsp90. Aim 1. Obtain three-dimensional structures for Hip and Hop, either alone or in co-crystals with Hsp70 or Hsp90. Aim 2. Test the hypothesis that the DP-repeat motifs appearing near the C-termini of both Hip and Hop play a major role in directing assembly progression of progesterone receptor complexes. The putative DP interaction sites on Hip and/or Hop will be defined by biochemical and molecular approaches. Next, mutant proteins will be generated to test the functional importance of these sites. Aim 3. Test the hypothesis that the Hsp70 partner BAG-1 is required for efficient assembly and hormone-dependent activation of PR complexes. Three different BAG1 isoforms will be compared for their abilities to participate during specific stages of progesterone receptor assembly, and participating isoforms will be tested for functional involvement in maintaining the dynamics of receptor-chaperone interactions and in promoting hormone-dependent dissociation of receptor complexes. Achieving these aims will provide a better basic understanding of the interplay between components of the molecular chaperone machinery. The knowledge gained from the steroid receptor model will also provide a paradigm for a variety of other signaling proteins whose activities are regulated by a similar chaperone pathway. One potential consequence of these studies is the development of novel therapeutic approaches to the treatment of steroid-dependent diseases and the modulation of normal steroid-mediated processes. However, since the pathway of chaperone interactions that underlies the maturation and maintenance of steroid receptors also occurs with proteins in other signal transduction pathways, there are many additional potential applications of these basic studies.